The adsorption of tobacco mosaic virus (TMV) on self-assembled and Langmuir-Blodgett monolayers was investigated using total internal reflection fluorescence (TIRF) spectroscopy and scanning force microscopy (SFM). Substrates were chosen to probe electrostatic, hydrophobic and surface fluidity effects on TMV adsorption. Positively charged and hydrophobic surfaces demonstrated similar initial rates of TMV adsorption; however, their respective surface TMV coverages differed greatly. Likewise, positively charged surfaces which differed primarily in surface fluidity exhibited similar adsorption rates for TMV, but different TMV surface coverages. In contrast, virus adsorption to negatively charged and zwitterionic substrates was negligible. To elucidate these differences in adsorption behavior, SFM was used to image the distribution and the aggregation state of adsorbed TMV.